Compound, 4-carboxyphthalato(1,2-diaminocyclohexane)-platinum(II) and alkali metal salts thereof

ABSTRACT

##STR1## 4-Carboxyphthalato (1,2-diaminocyclohexane)platinum(II) (and alkali metal salts thereof) has shown antileukemic activity in mice against murine leukemia L1210. It is effective in dosages of 5-60 mg/kg of body weight and is potentiated in a treatment with cyclophosphamide (CY) (50 mg/kg of body weight) to which may be added 5-fluorouracil (5-FU) (75 mg/kg of body weight) or hydroxyurea (HU) (1000 mg/kg of body weight). Some previous platinum chelate compounds show dosage limitation due to renal impairment but the free carboxyl of the present Pt compound offers a possible route of oral administration and absorption by the stomach. 
     The Pt compound may be prepared by reacting the known dichloro(1,2-diaminocyclohexane)platinum(II) (NSC 194814) with silver nitrate to replace chloro with nitro. Subsequently, benzene tricarboxylic acid is added to form an off-white precipitate (2 hrs, dark, 5° C) of the desired product which is preferably utilized as the alkali metal salt. 
     The 4-carboxyphthalato(1,2-diaminocyclohexane)-platinum(II) and alkali metal salts thereof may be combined in multiple drug regimen with substantially improved yield cures over the parent compound. For example, the compound denoted Pt-307 may be combined in a dual regimen with cyclophosphamide (CY) and in a triple drug regimen of Pt-307 plus cyclophosphamide (CY) and either 5-fluorouracil (5-FU) or hydroxyurea (HU) as the third component. ##STR2##

4-Carboxyphthalato (1,2-diaminocyclohexane) platinum (II) (and alkali metal salts thereof) has shown antileukemic activity in mice against murine leukemia L1210. It is effective in dosages of 5-60 mg/kg of body weight and is potentiated in a treatment with cyclophosphamide (CY) (50 mg/kg of body weight) to which may be added 5-fluorouracil (5-FU) (75 mg/kg of body weight) or hydroxyurea (HU) (1000 mg/kg of body weight). Some previous platinum chelate compounds show dosage limitation due to renal impairment but the free carboxyl of the present composition offers a possible route of oral injection and absorption by the stomach.

The compound may be prepared by reacting the known dichloro(1,2-diaminocyclohexane)platinum(II) (NSC 194814) with silver nitrate to replace chloro with nitro. Subsequently, benzene tricarboxylic acid is added to form an off-white precipitate (2 hrs, dark, 5° C.) of the desired product which is preferably utilized as the alkali metal salt.

The 4-carboxyphthalato(1,2-diaminocyclohexane)platinum(II) and alkali metal salts thereof may be combined in multiple drug regimen with substantially improved yield cures over the parent compound. For example, the compound denoted Pt-307 may be combined in a dual regimen with cyclophosphamide (CY) and in a triple drug regimen of Pt-307 plus cyclophosphamide (CY) and either 5-fluorouracil (5-FU) or hydroxyurea (HU) as the third component.

It is understood in this specification and claims that where the compound 4-carboxyphthalato(1,2-diaminocyclohexane)platinum(II) is utilized it is designed and meant to apply to the free acid as well as alkali metal salts thereof such as sodium, potassium, lithium, etc.

PRIOR ART

The following patents provide background material to the present invention.

U.S. Pat. No. 3,892,790 Tobe et al -- The compounds disclosed are platinum(II) halogeno complexes such as cis-dichloro-Pt(II) where the remaining bonds are bis(cyclopropylamine), bis(cyclobutylamine), bis(cyclopentylamine), bis(cyclohexylamine) and bis(cycloheptylamine).

U.S. Pat. No. 3,904,663 Tobe et al -- Formula 2 in the list of compounds bridging columns 1 and 2 is cyclohexane (trans)-1,2-diamine platinum(II) and the compounds for claim purposes are illustrated by dichloro(ortho-phenylene-diamine)platinum(II).

The following literature references are deemed of interest:

J. M. Hill et al, "Platinum Coordination Compounds in the Treatment of Acute Leukemia and Other Malignant Diseases with Particular Reference to Malonato 1,2-Diaminocyclohexane Platinum(II)," Journal of Clinical Hematology and Oncology, 7:681-697 (1977). This compound abbreviated PHM has a structure disclosed as follows: ##STR3##

Paul Schwartz et al, "Preparation and Antitumor Evaluation of Water-Soluble Derivatives of Dichloro(1,2-Diaminocyclohexane)Platinum(II) (NSC-194814)," accepted for publication in Cancer Treatment Reports.

Glen R. Gale et al, "Potentiating Action of 5-Fluorouracil When Used in Combination with Platinum Compounds and Cyclophosphamide in Treatment of Advanced L1210 Leukemia," accepted for publication in Bioinorganic Chemistry.

Glen R. Gale et al, "Synergistic Action of High-Dose Hydroxyurea When Used With Cyclophosphamide and Certain New Organoplatinum Complexes in Treatment of Advanced L1210 Leukemia," accepted for publication in Cancer.

The utilization of the present compound, 4-carboxyphthalato(1,2-diaminocyclohexane)platinum(II), against L1210 murine leukemia is not anticipated by the effective prior art and it is noted that the last three literature references, one to Schwartz et al and two to Gale et al, as yet unpublished, are useful in the preparation of the present specification of invention.

The recent chemotherapy of utilizing platinum coordination compounds dates back to some work by Rosenberg and co-workers as reported in Nature, 205:698-699 (1965) and also in Nature, 222:385-386 (1969). It was early found that the cis platinous compounds had superior activity and as a starting point was mentioned cis-dichloro(1,2-diaminocyclohexane)platinum(II) known as DDCP and cis-platinous diaminodichloride (PDD). In general, newer antitumor agents modified or replaced the chlorides in dichloro-1,2-diaminocyclohexane platinum(II) (NSC 194814) by organic or inorganic anion. One difficulty with the diaminocyclohexane complex is its low solubility. It was known that the diaminocyclohexane moiety reduces the toxicity while increasing the antitumor activity of complexes with highly reactive ligands as, for example, nitrato or sulfato groups.

In the present application a dicarboxylic acid dianion (the phthalate) relates to the central platinum(II) cation and forms neutral complexes.

Additionally, while phthalato(1,2-diaminocyclohexane)platinum(II) (NSC 268255) is very insoluble, the 4-carboxyphthalato(1,2,-diaminocyclohexane)platinum(II) (NSC 271674) readily dissolves in 1% sodium bicarbonate solution via formation of the sodium carboxylate. This characteristic of the present compound is quite important since it not only makes administration in clinical trials possible but leads to the possibility of oral ingestion later in humans.

The presence of a carboxylate group on the molecule suggests the possibility that the compound may be absorbed from the stomach (low pH, suppression of dissociation of proton). This characteristic would be decidedly advantageous in that effective blood levels could be achieved by oral administration instead of the more cumbersome intravenous route to humans.

PREPARATION OF COMPOUND 4-CARBOXYPHTHALATO(1,2-DIAMINOCYCLOHEXANE)PLATINUM(II)

4-Carboxyphthalato(1,2-diaminocyclohexane)platinum(II) has the following structure: ##STR4## Molecular formula: C₁₅ H₁₈ N₂ O₆ Pt Molecular weight: 517.44

This compound may be prepared by the following synthetic route: ##STR5##

A specific preparation is set out post as Example 1.

DOSAGE

Compound Pt-307 was administered usually in a dosage regimen of 5-60 mg/kg of body weight. This was given as a single dose or on Days 1, 5, and 9 in the standard NIH 10-day testing. In this testing Pt-307 was the same compound as NSC 271674. In general, the ternary combination such as Pt + CY + FU enhanced markedly the increased lifespan of treated mice. Collectively the cure rate (greater than 60-day survival) was less than 6% with the Pt + CY combinations and was increased to over 63% upon inclusion of FU in the regimen. This particular combination was somewhat superior to that obtained with hydroxyurea (HU) below where HU was used as a third drug with the Pt + CY combination.

It is noted that 5-fluorouracil (5-FU) has also been shown to potentiate the action of irradiation. For example, mean survival times were significantly increased in patients who received combined FU and radiation treatment as compared to radiation therapy alone for adenocarcinoma of the stomach, pancreas, and large bowel. The Pt + CY + FU combinations proved to be uniquely efficaceous against advanced L1210 leukemia and specially in combination chemotherapy. CY was utilized at an optimum 50 mg/kg of body weight (range 25-75 mg/kg) preferably given at Day 3 in the 10-day testing. 5-FU was utilized at 75 mg/kg of body weight (range 50-100 mg/kg) at Day 3. HU was used in a high dosage of at least 1000 mg/kg of body weight (range 1000-1500 mg/kg). The 5-FU and the HU were utilized as ternary compositions only with cyclophosphamide and the platinum compound.

HIGH-DOSE HYDROXYUREA USED WITH CYCLOPHOSPHAMIDE AND PT NSC 271674 (Pt-307)

Hydroxyurea is utilized in combination therapy at a high dosage of at least 1000 mg/kg of body weight (range 1000-1500 mg/kg). At this dosage when used alone it is only minimally effective. In triple drug regimen involving Pt + CY and the hydroxyurea (HU), the cure rate is 11% with the dual combinations and is increased to 53% upon inclusion of HU in the regimen. It is believed that HU may inhibit a process whereby potentially lethal DNA damage produced by Pt + CY would otherwise be repaired.

EXAMPLE 1

Dichloro(1,2-diaminocyclohexane)platinum(II) (19.53 g; 0.052 mole) was suspended in 300 ml of water. Silver nitrate (15.20 g; 0.085 mole) was added and the mixture was stirred in a darkened flask overnight. The mixture was filtered and the filtrate was added to 9.90 grams of 4-carboxyphthalic acid (1,2,4-benzenetricarboxylic acid) (0.047 mole) dissolved in 200 ml of warm water. The reaction mixture was kept in a darkened flask at room temperature for two hours and then stored at 5° C. overnight. The off-white precipitate was collected, washed thoroughly with water and dried in vacuo over Drierite to yield 12.0 g (55%) of 4-carboxyphthalato (1,2-diaminocyclohexane)platinum(II).

Elemental analyses (Galbraith Laboratories, Knoxville, Tennessee):

    ______________________________________                                                    % Calculated  % Found                                               ______________________________________                                         C            34.81           34.50                                             H            3.48            3.85                                              Pt           37.70           37.08                                             ______________________________________                                    

The compound was insoluble in water but dissolved readily in 1% sodium bicarbonate solution with the evolution of carbon dioxide indicating the presence of a free carboxy group. It was also very soluble in dimethylsulfoxide (DMSO). It decomposed without melting at temperatures greater than 270° C.

Thin layer chromatography, with methanol as developer, of a DMSO solution (dissociation was suppressed by the addition of 1,2,4-benzene-tricarboxylic acid to the solution) on an Eastman 13255 cellulose chromagram sheet gave an R_(F) value of 0.75. Stannous chloride solution was the detecting agent.

The infrared spectrum of a KBr pellet gave absorbances at the following wavenumbers (cm⁻¹):

    ______________________________________                                         3450s,                                                                               3210s,  3100w,    2935s,                                                                               2860m,                                                                               2600w, 1720s,                              1600s,                                                                               1490s,  1450m,    1350s,                                                                               1240m,                                                                               1160m, 1120m,                              1090w,                                                                               1060s,  1028vw,   1025m,                                                                               980w, 915m,  860w,                               840m, 800m,   760s,   700m, 670vw,                                                                               655w, 620m, 570                                                                            vw,                              510m, 440m,   360m,   305w.                                                    ______________________________________                                    

EXAMPLE 2 Effective Dosage in Mice

The compound in 1% NaHCO₃ solution was tested against the L1210 leukemia in BDF₁ mice [10⁶ cells given to mice on Day 0, treatment (ip) on Day 1 only] in laboratory and the following % increases in life span (% ILS) were noted:

    ______________________________________                                         Dose, mg/kg     % ILS                                                          ______________________________________                                          5               70                                                            10              118                                                            20              119                                                            25              139                                                            40              232                                                            50              288 (1 of 8 mice survived                                                      >60 days)                                                      60              174 (1 of 8 mice survived                                                      >60 days)                                                      ______________________________________                                    

The compound singly and in combination regimen

When treatment was delayed until Day 3, 5 mg/kg gave 51% ILS, 42% ILS and 39% ILS in three experiments. In combination with cyclophosphamide (50 mg/kg) in Day 3 treatment, 5 mg/kg gave 416% ILS with 3 of 10 >60 day survivors and 217% ILS with no >60 day survivors in two experiments. In combination with cyclophosphamide (50 mg/kg) and 5-fluorouracil (75 mg/kg) in Day 3 treatment, 5 mg/kg gave 673% ILS with 8 of 10 >60 day survivors.

The approximate LD₅₀ was 75 mg/kg.

The compound was submitted to the Drug Development Branch of the National Cancer Institute and assigned NSC No. 271674. It was screened against the L1210 leukemia on a day 1, 5, 9 schedule and the following results were obtained:

    ______________________________________                                         Dose, mg/kg    % ILS       Cures (of 10)                                       ______________________________________                                         25             344         5                                                   12.5           191         2                                                   6.25           163         1                                                   3.13            46         0                                                   1.56            33         0                                                   0.78            27         0                                                   ______________________________________                                    

A second screener obtained:

    ______________________________________                                         Dose, mg/kg % ILS                                                              ______________________________________                                         25          86                                                                 12.5        147                                                                6.25        58       (2 questionably toxic deaths                                                   were noted)                                               3.13        41                                                                 1.56        19                                                                 0.78        19                                                                 ______________________________________                                    

No cures were obtained by the second screener. 

We claim:
 1. ##STR6## 4-carboxyphthalato(1,2-diaminocyclohexane)platinum(II) and alkali metal salts thereof. 